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Open Access

Methodology article

Optimization of immunomagnetic separation for cord

blood-derived hematopoietic stem cells

Tuija Kekarainen

†1,2

, Sirkka Mannelin

1

, Jarmo Laine

1

and Taina Jaatinen*

†1

Address: 1Finnish Red Cross Blood Service, Helsinki, Finland and 2Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de

Barcelona, Barcelona, Spain

Email: Tuija Kekarainen - tuija.kekarainen@cresa.uab.es; Sirkka Mannelin - sirkka.mannelin@bts.redcross.fi; Jarmo Laine - jarmo.laine@bts.redcross.fi; Taina Jaatinen* - taina.jaatinen@bts.redcross.fi

* Corresponding author †Equal contributors

Abstract

Background: There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood.

Results: In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin-cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential.

Conclusion: The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood.

Background

Hematopoietic stem cells (HSC), with their unique self-renewal and differentiation capacity, offer great potential for the treatment of hematological disorders, immunode-ficiency and inborn errors of metabolism [1,2]. HSCs can be collected from mobilized peripheral blood (PB), bone marrow (BM) and cord blood (CB). Lately, CB has been increasingly utilized because it is readily available, HLA mismatches are better tolerated and there is a decreased risk of graft-versus-host disease when using CB-derived HSCs when compared to the other sources [3]. Even though the cell content of CB is limited, it has a higher fre-quency of progenitor cells compared to PB or BM [3-5]. CB-derived CD34+ cells have also been shown to

prolifer-ate more rapidly than their counterparts from BM [6], and CB-derived HSCs possess increased engraftment potential when compared to cells from PB or BM [7,8]. In addition, recent studies suggest that CB is a source of non-hemat-opoietic stem or progenitor cells, such as mesenchymal and endothelial precursors [9,10].

Enrichment of HSCs is based on the expression of certain surface antigens or on the lack of expression of lineage-specific antigens. The most commonly used surface marker for HSC selection is the transmembrane glycopro-tein CD34. CD34 is also used to quantify the stem cell content of CB units banked for clinical use [11]. Most, if not all, CD34+ cells express the CD133 glycoprotein on

Published: 01 August 2006

BMC Cell Biology 2006, 7:30 doi:10.1186/1471-2121-7-30

Received: 03 November 2005 Accepted: 01 August 2006 This article is available from: http://www.biomedcentral.com/1471-2121/7/30

© 2006 Kekarainen et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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their surface. CD133 appears to be expressed on more primitive cells and CD133+ cell grafts have been tested in stem cell transplantation [12-14]. Alternative, but cur-rently poorly characterized Lin- progenitor cells lack line-age-specific markers [15,16]. Fluorescence-activated cell sorting and immunomagnetic selection systems utilize antibodies against these cell surface antigens to enrich HSCs. However, the major challenge has been the diffi-culty to produce highly pure HSC fractions from CB with good recovery. Furthermore, the handling of CB is chal-lenging due to the relatively high content of thrombocytes and nucleated erythroid precursors which have a negative impact on the mononuclear cell isolation. For these rea-sons, standardized protocols for PB sample handling and cell separation do not work well for CB. Only few studies have investigated the efficiency of the immunomagnetic selection method used to isolate CD34+ cells from CB. Belvedere et al. compared the results from 49 selections and reported mean CD34+ cell purities of 41% and 85% after first and second passage through the separation col-umn, respectively [17]. Melnik et al. report an average purity of 60% for CB-derived CD34+ cells from 10 separa-tions [18].

In this study, three different protocols were optimized to enrich CD34+, CD133+ and Lin- HSCs with over 90% purity from both fresh and cryopreserved CB. Cryopre-served CB cells have been considered to be especially chal-lenging in selection procedures because of cell aggregation caused by cell damage during thawing. The used protocols were based on positive selection of cells expressing CD34

and CD133, or on depletion of cells expressing lineage-specific markers. The magnetic cell sorting system MACS was used due to its gentleness and time-effectiveness. Fur-ther, the clonogenic capacity of selected HSC populations was determined using the colony-forming unit (CFU) assay.

Results and discussion

Handling of CB cells

The isolation of pure mononuclear cell (MNC) fractions from CB, and subpopulations thereof, brings about a spe-cial challenge. This appears to be due to the large number of thrombocytes and erythroid progenitors in CB. In the Ficoll-Paque density gradient, all erythroid cells do not necessarily sediment to the bottom layer, but are retained in the interphase of plasma and Ficoll-Paque. The eryth-roid cells remaining in the interphase are nucleated pro-genitors that may hamper the subsequent immunomagnetic selection of HSC populations. Treat-ment with ammonium chloride or diethylene glycol may be used to deplete red blood cells, but depletion was not performed in this study as the nucleated erythroid progen-itors are not easily lyzed and purities up to 97% were reached without any additional treatment. The unusually slow sedimentation of erythroid cells is not seen when working with PB.

When handling cryopreserved CB cells, aggregation was observed. Aggregation was reduced by replacing ethylene-diamine tetraacetic acid (2 mM EDTA, Merck, Darmstadt, Germany) with anticoagulant citrate dextrose solution, formula A (0.6% ACD/A, Baxter Healthcare, Lessines, Bel-gium) in the sample buffer. In some cases aggregation was so substantial that the cells needed to be resuspended in DNaseI containing buffer. DNaseI digests the DNA released from dead cells and prevents aggregation. The DNase treatment did not affect the viability or colony-forming potential of selected CB cells. No cell aggregation was seen when handling fresh CB cells.

MNC fraction

In cryopreserved CB, the mean MNC concentration was 5.14 × 109/l (range 0.96–10.00, SD = 3.43) (Figure 1), and the mean platelet concentration was 8.30 × 109/l (range 0.00–17.00, SD = 5.46). Cryopreserved CB contained a mean of 0.14 × 1012/l erythrocytes (range 0.02–0.67, SD = 0.20), and the mean hematocrit was 2%.

Fresh CB contained a mean of 2.68 × 109/l MNCs (range 1.24–3.62, SD = 0.64) (Figure 1). The difference in MNC concentration between cryopreserved and fresh samples was not statistically significant (P = 0.06). The remarkably high disparity in the standard deviation of MNC concen-tration between cryopreserved and fresh CB may be due to the processing and freezing of cells performed to bank the

Mean MNC count in cryopreserved and fresh CB Figure 1

Mean MNC count in cryopreserved and fresh CB. The

mean MNC concentration for cryopreserved and fresh CB was 5.14 × 109/l (range 0.96–10.00, median 4.30, SD = 3.43)

and 2.68 × 109/l (range 1.24–3.62, median 2.77, SD = 0.64),

respectively. The difference between cryopreserved and fresh CB was not statistically significant (P = 0.06). Bars show means and error bars show standard deviations. Abbrevia-tions: MNC, mononuclear cells; CB, cord blood.

0 2 4 6 8 10 Cryopreserved CB Fresh CB Tot al M N C c o unt x 1 0 9/l n=10 n=9

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CB units [19]. The mean concentration was 205.89 × 109/ l (range 84.00–505.00, SD = 130.58) for platelets and 0.12 × 1012/l (range 0.03–0.50, SD = 0.14) for erythro-cytes. The mean hematocrit was 1%.

Immunomagnetic separation of HSC populations

When using the Direct CD34 Progenitor Cell Isolation Kit with single column separation and the labeling protocol recommended by the manufacturer (Miltenyi Biotec, Ber-gisch Gladbach, Germany), a purity of less than 50% was reached for the CD34+ cells (Figure 2). To obtain highly pure CD34+ cells, the immunomagnetic selection method was optimized. Several washing steps (3–10) were tested for single column separation. A purity of 80% was achieved with extensive washing, but the yield was poor (less than 50% of the expected yield). Two successive col-umn separations resulted in 77% purity, but a great number of CD34+ cells were still lost during the process indicating a further need to optimize the protocol. An additional labeling step between the two column separa-tions increased the purity to >90% (results for a represent-ative sample shown in figure 3) and resulted in an acceptable yield as well. The optimized two-column method with additional labeling proved reliable and was applied to the separation of both CD34+ and CD133+ cells. The average yield of CD34+ and CD133+ cells from

one cord blood unit was 106 and 105, respectively. The purity of positively selected CD34+/CD133+ cells was reproducibly over 90% and their negative counterparts were nearly 100% pure.

Generally, the recovery of CD34+ and CD133+ cells was 0.86% (range 0.56–1.45, SD = 0.36) and 0.21% (range 0.04–0.41, SD = 0.12), respectively. The recovery of CD34+ cells was higher from fresh CB (0.97%) when compared to cryopreserved CB (0.78%), although the dif-ference was not statistically significant (P= 0.54). The results are consistent with the study by Almici et al. show-ing no significant difference in yield or in purity for fresh CB CD34+ cells in comparison to crypreserved cells [20]. This was the case with CD133+ cells as well, the recovery being 0.29% for fresh CB and 0.12% for cryopreserved CB (P = 0.11). The purities were not affected by the initial per-centage of HSC populations in CB. The results of the purity assessment for representative samples of CD34+/-, CD133+/- and Lin-/+ cells are shown in figure 4.

The magnetic sorting of Lin- cells was optimized to find the optimum concentrations for antibodies and magnetic colloids. Lin- cells were separated from the MNC fraction with magnetic cell sorting and the purity of the enriched cell fraction was analyzed by flow cytometry. The average

Purity assessment of the CD34+ cell fraction by flow cytometry Figure 2

Purity assessment of the CD34+ cell fraction by flow cytometry. The initial purity of CD34+ cells after separation through single column was 47.5%. The CD34- fraction was 99.4% pure. CD34+ and CD34- cell populations were defined by first gating on forward and side scatter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody. Percentages indicating the purity of isolated cell fractions are shown for both plots. Abbreviations: SSC, side scatter; PE, phycoerythrin.

CD34+ 47.5%

CD34- 99.4%

SSC

-H

eig

h

t

CD34 PE

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yield of Lin- cells from one cord blood unit was 106. The overall recovery of Lin- cells was 0.29% (range 0.13–0.70, SD = 0.21), being higher in fresh CB samples (0.43%) than in cryopreserved CB samples (0.16%). The difference between fresh and cryopreserved samples was not statisti-cally significant (P = 0.19). The mean purity of Lin- cell population was 90% (range 82–98%, SD = 5.00) and it did not differ between fresh and cryopreserved CB units. To assess the effect of antibody concentration on the selec-tion of Lin- cells, varying amounts of the antibody cocktail (50–100 µl/ml) and magnetic colloids (30–60 µl/ml) were tested. The concentration of antibodies and mag-netic colloids did not affect the purity of Lin- cells based on flow cytometric analysis.

All the optimized protocols are described in detail in fig-ure 5. The operation time for the selection of CD34+/- and CD133+/- cells using MiniMACS or MidiMACS is approx-imately 1.5 hours. The operation time for isolation of Lin-/+ cells is approximately 45 minutes. The selection can be made more effective with the AutoMACS system devel-oped for high-speed automated cell sorting. The opti-mized protocols have been developed for enrichment of CB HSCs for research purposes. However, the enrichment of stem and progenitor cells is often necessary in clinical settings. The selected HSCs are increasingly used in

trans-plantations and enrichment may be required for deple-tion of contaminating mature cells or tumor cells. Potentially, the methods described here could be applied to clinical-grade selection using the CliniMACS System that is CE-marked for clinical use in Europe.

With the optimized protocols, a purity of at least 90% was achieved for CD34+, CD133+ and Lin- cells. Viability was 99% for all the selected cell types. This demonstrates that the optimized protocols work well for HSC enrichment from both fresh and cryopreserved CB. Fresh CB was eas-ier to handle and the recovery of HSCs was higher from fresh CB. Nonetheless, cryopreserved cord blood is almost exclusively used in clinical settings. Therefore, if one wishes to use selected HSCs, the cells should preferably be isolated on fresh cord blood and cryopreserved after the selection procedure to maximize the recovery of HSCs. HSCs, enriched by the protocols described here, have been used in gene expression studies with great reproduc-ibility and consistency [21].

It has been suggested that the binding of an antibody to the surface of a HSC may influence cell proliferation and differentiation by activating intracellular signaling path-ways [14]. An anti-CD34 antibody has been shown to induce tyrosine phosphorylation in BM-derived CD34+

Purity assessment of CD34+ cell fraction after one or two column separations Figure 3

Purity assessment of CD34+ cell fraction after one or two column separations. A) The CD34+ cell fraction was 78% pure after the first column separation. B) A 92% pure CD34+ cell faction was obtained by an additional labeling step in connec-tion with a second column separaconnec-tion. CD34+ cell populaconnec-tions were defined by first gating on forward and side scatter proper-ties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody. Percentages indicating the purity of isolated cell fractions are shown for both plots. Abbreviations: SSC, side scatter; PE, phycoerythrin.

CD34+ 78%

CD34+ 92%

A

B

SSC

-H

eig

h

t

CD34 PE

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Purity assessment of CD34+/-, CD133+/- and Lin-/+ cell fractions Figure 4

Purity assessment of CD34+/-, CD133+/- and Lin-/+ cell fractions. A) Purities for CD34+ and CD34- cell factions were 97.1% and 99.1%, respectively. B) Purities for CD133+ and CD133- fractions were 93.6% and 99.1%, respectively. C) Purities for Lin- and Lin+ cell factions were 97.0% and 99.5%, respectively. CD34+/-, CD133+/- cell populations were defined by first gating on forward and side scatter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody. Percentages indicating the purity of isolated cell fractions are shown for both plots. Abbreviations: SSC, side scatter; IgG, immunoglobulin; PE, phycoerythrin.

CD133+ 93.6% CD133- 99.1% CD34+ 97.1% CD34- 99.1% A B Lin- 97.0% Lin+ 99.5% C S S C-Height CD34 PE SSC -H eig h t SSC -H eig h t CD133 PE anti-mouse IgG PE

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A chart of the optimized protocols to isolate CD34+/-, CD133+/- and Lin-/+ cells from cord blood Figure 5

A chart of the optimized protocols to isolate CD34+/-, CD133+/- and Lin-/+ cells from cord blood. Isolation of CD34/- and CD133+/- cells was performed using Direct CD34 Progenitor Cell Isolation Kit (#130-046-702, Miltenyi Biotec) and CD133 Cell Isolation Kit (#130-050-801, Miltenyi Biotec), respectively. Lin-/+ cells were isolated using StemSep Human Progenitor Enrichment Kit (#14056, StemCell Technologies). For all magnetic separations, MACS columns and separators (Miltenyi Biotech) were used. Abbreviations: MNC, mononuclear cells; Buffer, PBS pH 7.2 supplemented with 0.5% bovine serum albumin and 2 mM EDTA or 0.6% ACD/A.

MNC

1. labeling

Prepare cell suspension of 300 µl of Buffer per 108MNCs

Add 100 µl FcR Blocking Reagent and 100 µl Microbeads per 108MNCs Incubate at 6°C for 30 min

Wash with 10 ml of Buffer

Centrifuge for 10 min at 300xg at 20°C Resuspend in 500 µl of Buffer per 108MNCs

1. magnetic separation

Choose column according to the number of MNCs

MS for < 2 x 108MNCs / LS for 2 x 108– 2 x 109MNCs Place column in the magnetic field of the MACS separator Rinse column with Buffer

500 µl MS / 3 ml LS

Apply labeled cell suspension to the column

Collect flowthrough as the CD34-/CD133- cell fraction Wash column with Buffer

4 x 500 µl MS / 4 x 3 ml LS

Remove column from magnetic field and place on a tube Elute and collect CD34+/CD133+ cells with Buffer

2 x 1 ml MS / 2 x 5 ml LS

(use the plunger supplied with the column on the second elution)

2. labeling

Centrifuge for 5 min at 300xg at 20°C Resuspend in 300 µl of Buffer

Add 25 µl FcR Blocking Reagent and 25 µl Microbeads Incubate at 6°C for 15 min

Wash with 4 ml of Buffer

Centrifuge for 5 min at 300xg at 20°C Resuspend in 500 µl of Buffer

2. magnetic separation

Place MS column in the magnetic field of the MACS separator Rinse column with 500 µl of Buffer

Apply labeled cell suspension to the column Wash column with Buffer 4 x 500 µl

Remove column from magnetic field and place on a tube Elute and collect CD34+/CD133+ cells with Buffer 2 x 500 µµµµl (use the plunger supplied with the column on the second elution)

Labeling

Prepare cell suspension of 1 ml of Buffer per 8 x 107MNCs Add 100 µl Progenitor Enrichment Cocktail

Incubate at RT for 15 min Add 60 µl Magnetic Iron Particles Incubate at RT for 15 min

Magnetic separation

Place LD column in the magnetic field of the MACS separator Rinse column with 2 ml of Buffer

Apply labeled cell suspension to the column Collect flowthrough as the Lin- cell fraction Wash column with Buffer 2 x 1 ml

(collect in the same tube with flowthrough)

Remove column from magnetic field and place on a tube Elute and collect Lin+ cells with Buffer 2 x 1 ml

(use the plunger supplied with the column on the second elution) CD34+/- and CD133+/- cells Lin-/+ cells

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cells [22]. However, Lin- cells are selected through nega-tive depletion. Thus, neither antibody binding nor activa-tion of signaling pathways is expected. Further studies on the effect of the interaction between HSCs and the anti-bodies used for their selection as well as the possible impact of this contact on HSC quality are awaited. Low number of HSCs in cord blood is a limitation for its use. Ex vivo expansion of HSCs may be used to generate the clinically relevant cell numbers needed for adult patients. However, mature cells may develop during long-term culture and result in a need for reselection of progen-itor cells. The optimized protocols can be applied for enrichment of stem and progenitor cells after ex vivo

expansion.

Colony forming unit assay

The CFU assay was used to measure the clonogenic capac-ity of CD34+, CD133+ and Lin- cells as well as MNCs. Total CFU (CFU-TOT) number was determined as the sum of granulocyte-erythroid-macrophage-megakaryo-cyte (CFU-GEMM), granulogranulocyte-erythroid-macrophage-megakaryo-cyte-macrophage (CFU-GM), erythroid (CFU-E) and burst-forming erythroid (BFU-E) colonies. CFU-TOT counts were 84.5, 80.0, 57.3 and 0.5 per 1000 cells for CD34+, CD133+, Lin- and MNCs, respectively. CD34- and CD133- cell populations have shown very limited colony forming potential in our previ-ous studies with CFU-TOT counts of 0.1 and 0.6 per 1000 cells, respectively.

CFU-GM colonies (mean 53.2%) and CFU-GEMM colo-nies (mean 32.6%) were the most common colony types formed by of CB-derived HSCs. The proportion of differ-ent colony types for CD34+, CD133+, Lin- and MNCs is shown in Table 1. BFU-Es represented a mean of 12.9% of the colony content of HSCs. However, the Lin- cell frac-tion formed a surprisingly large number of BFU-Es (27.3%), probably due to the inefficient removal of eryth-roid progenitors during the depletion procedure. Very lit-tle CFU-E colonies were observed (mean 1.3%), MNC population being the most efficient in forming them (5.5%). The high proportion of BFU-E and CFU-E colo-nies formed by the MNC population reflects the unusual sedimentation of erythroid progenitors in Ficoll-Paque density gradient. The results show that all the selected

HSC populations have substantial clonogenic potential and are highly non-committed. Taken together, the data support the use of traditionally used markers to separate HSC populations until more specific markers are found.

Conclusion

Immunomagnetic cell sorting enables fast and gentle sep-aration of HSCs. However, the previously reported proto-cols are not optimal for CB and result in unsatisfactory purity and yield, indicating a need for optimization of the procedures. With the modified protocols presented here, over 90% pure HSC fractions can be reproducibly obtained. This is essential for the use of specific hemat-opoietic progenitor cell types in research and therapeutic applications.

The single most important factor influencing engraftment in HSC transplantation appears to be the nucleated cell content. Even though the cell content is limited in CB and there is no possibility to obtain an additional graft from the same donor, the increased engraftment potential of CB-derived HSCs makes them an appealing alternative for HSCs from PB or BM. It remains to be seen whether the total nucleated cell content or a population of highly pure and specific hematopoietic progenitor cells will prove to be more important for graft potency.

Methods

CB units

Umbilical CB was obtained from informed and consent-ing donors at the Helsinki University Central Hospital, Department of Obstetrics and Gynaecology. Permit to col-lect and use donated stem cells has been obtained from the ethics board of the Helsinki University Central Hospi-tal (550/E8/02) and the ethics board of the Finnish Red Cross Blood Service (40/02).

The umbilical cord was clamped according to standard hospital procedure and CB collections were performed ex utero. CB was collected into a sterile collection bag (Cord Blood Collection system, Medsep Corporation, Covina, USA) containing 25 ml of Citrate Phosphate Dextrose solution. The collection volume varied between 45–105 ml. Some CB units were volume reduced and cryopre-served in a BioArchive system at the Finnish Red Cross Blood Service, Cord Blood Bank as previously described [16,19] and some units were processed freshly within hours from collection. Altogether, 8 cryopreserved and 12 fresh CB units were used to optimize the protocols. In addition, 10 cryopreserved and 9 fresh CB units were used to test the optimized protocols.

Handling of CB units

Cryopreserved CB unit was taken from the BioArchive sys-tem and kept for 2 minutes in the gas phase of liquid

Table 1: Frequency of different types of CFU colonies within CD34+, CD133+, Lin- and MNC populations.

Cell type CFU-GM CFU-GEMM BFU-E CFU-E

CD34+ 58.2% 33.2% 7.2% 1.4%

CD133+ 57.4% 38.3% 4.3% 0.0%

Lin- 43.8% 26.3% 27.3% 2.6%

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nitrogen, 3–5 minutes at room temperature and then in 37°C water bath until completely thawed. CB was trans-ferred from the freezing bag into 50 ml tubes containing 10 ml of freezing solution: 2.5% albumin (Finnish Red Cross, Blood Service, Helsinki, Finland), 50% NaCl (Bax-ter Healthcare) and 50% Gentran40 (Bax(Bax-ter Healthcare). Cells were pelleted by centrifugation at 600 × g for 10 minutes and the supernatant was discarded. When cell aggregation occurred, the cell pellet was resuspended in 200 µl of 1 mg/ml DNaseI (Sigma-Aldrich, Steinheim, Germany). Cells were then suspended carefully in 100 ml of phosphate-buffered saline (PBS, pH 7.4) supplemented with 0.6% ACD/A. Fresh CB was diluted 1:4 with PBS sup-plemented with 2 mM EDTA, to reduce the size and number of cell aggregates and to give better lymphocyte yield in density gradient centrifugation.

MNCs were isolated by density gradient using 15 ml of Ficoll-Paque reagent (Amersham Biociences, Piscataway, USA) and 35 ml of diluted CB. The two-phase system was centrifuged at 400 × g for 40 minutes. MNCs, collected from the interface of the two phases, were washed twice with PBS. MNC counting was performed by automatic cell counter Sysmex K-1000 (Sysmex Corporation, Kobe, Japan).

Separation of CD34+/CD133+ cells

CD34+ and CD133+ cells were enriched through positive selection using MiniMACS or MidiMACS separation sys-tems (Miltenyi Biotec). For the labeling of CD34+ cells, Direct CD34 Progenitor Cell Isolation Kit was used, whereas CD133+ cells were labeled using the CD133 Cell Isolation Kit. 100 µl of FcR Blocking Reagent, to inhibit unspecific or Fc-receptor mediated binding, and 100 µl of CD34/CD133 MicroBeads for magnetic labeling of cells were added per 108 cells, as per manufacturer's recom-mendations.

MS or LS MACS affinity columns were used depending on the number of MNCs. MS column was used for up to 2 × 108 MNCs, and LS column was used for 2 × 108 to 2 × 109 of MNCs. Labeled cell suspension was subjected to immu-nomagnetic separation where magnetically labeled cells retain in the column while the unlabeled cells pass through the column. After several washes, the column was removed from the magnet and the retained CD34+ or CD133+ cells were eluted with 1–5 ml of PBS supple-mented with 0.5% bovine serum albumin and 2 mM EDTA. CD34+ and CD133+ cells were subjected to one or two rounds of separation and their negative counterparts were collected for control purposes. In the two-column system, an additional labeling step between the column separations was tested, using 25 µl of both FcR Blocking Reagent and MicroBeads. The optimum purity and yield

was obtained when using the additional labeling step in connection with the two-column system.

Separation of Lin- cells

To enrich progenitor cells, lineage committed cells were depleted. MNCs (8 × 107/ml) were labeled with Progeni-tor Enrichment Cocktail containing antibodies against CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b and Glycophorin A (StemCell Technologies, Vancouver, Canada) at room temperature for 15 minutes. Subse-quently, the cell suspension was incubated with magnetic iron particles at room temperature for 15 minutes. Vary-ing concentrations of StemSep Progenitor Enrichment Cocktail (100 µl/ml, 75 µl/ml, and 50 µl/ml) and mag-netic iron particles (60 µl/m, 45 µl/ml, and 30 µl/ml) were tested. Cell suspension was loaded into MACS LD column (Miltenyi Biotec) and unlabeled cells passing through the column were collected (Lin- fraction). The column was then washed twice with 1 ml of buffer and the remaining Lin+ cells were collected for control purposes. The conditions resulting in optimal separation of Lin-cells were 100 µl of Progenitor Enrichment Cocktail and 60 µl of magnetic iron particles per 8 × 107/ml MNCs, as recommended by the manufacturer.

Purity

To determine the purity of CD34+/- and CD133+/- cell fractions, 1 × 105 cells were labeled with fluorescein iso-thiocyanate (FITC)-conjugated anti-CD45 (clone 2D1, Becton Dickinson, Franklin Lakes, USA) and phycoeryth-rin (PE)-conjugated anti-CD34 (clone 345802 Becton Dickinson) or PE-conjugated anti-CD133 (clone 293C3, Miltenyi Biotec) monoclonal antibodies at 4°C for 15 minutes. Lin-/+ cells were labeled with PE-conjugated mouse immunoglobulin specific polyclonal anti-body (BD Biosciences, San Jose, USA). Platelets were detected with PE-conjugated anti-CD41a monoclonal antibody (BD Biosciences, San Jose, USA). Isotype-identi-cal monoclonal antibodies served as controls. Labeled cells were analyzed using Becton Dickinson FACSCali-bur™ with a 488 nm blue argon laser. Fluorescence was measured using 530/30 nm (FITC) and 585/42 nm (PE) bandpass filters. Data were analyzed using the Pro-COUNT™ software (BD Biosciences) or Windows Multi-ple Document Interface for Flow Cytometry, WinMDI version 2.8 [23]. CD34+, CD133+ and Lin- cell popula-tions were defined by first gating on forward and side scat-ter properties excluding platelets and debris. Subsequent gates were set to exclude >99% of control cells labeled with isotype-specific antibody.

Colony forming unit assay

MNCs (1 × 105) and enriched HSCs (2 × 103) suspended in 300 µl Iscove's Modified Dulbecco's Medium supple-mented with 2% fetal bovine serum (Gibco/Invitrogen,

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Paisley, United Kingdom) were mixed vigorously with 3 ml of MethoCult GF H4434 containing recombinant cytokines and erythropoietin (StemCell Technologies). The cells in MethoCult medium were plated in duplicate into sterile 35 mm petri dishes, and colonies were scored according to their morphological characteristics by light microscopy after a 14-day culture. Cell culture and colony scoring were performed according to accredited method-ology (Finnish Cord Blood Bank, Finnish Red Cross Blood Service, Helsinki, Finland) following international standards [24,25]. The CFU assay was performed in tripli-cate for each cell type (CD34+, CD133+, Lin- and MNC). Statistical analysis

For statistical analysis of total MNC counts between cryo-preserved and fresh CB samples, the non-parametric Mann-Whitney test was used, as the MNC counts were not normally distributed. The two groups, cryopreserved and fresh CB, were considered independent as they have been produced using different protocols. The recovery of CD34+, CD133+ and Lin- cells between cryopreserved and fresh CB was compared in independent samples by the t test and equality of variances was checked by Lev-ene's test. When the assumption of equality was not satis-fied, as with Lin- cells, the t test for non-equal variances was used to compensate the lack of homoscedasticity. P values less than 0.05 were considered statistically signifi-cant. The analyses were performed by Microsoft Excel and SPSS 12.0.1.

Authors' contributions

TK and TJ contributed equally to the work and partici-pated in the design of the study, optimized the experimen-tal procedures, performed the mononuclear cell fractionation, HSC selections and flow cytometric analy-ses, and drafted the manuscript. SM performed the CFU assays and participated in the interpretation of data. JL participated in the design and coordination of the study, and assisted in drafting the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are very grateful to Finnish Red Cross Blood Service Cord Blood Bank for providing the cord blood samples. We thank Hanna Salo for assistance with statistical analyses and Heidi Hemmoranta for helpful comments on the manuscript.

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References

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